Project 2 Summary Tobacco smoke (TS) is a major cause of bladder cancer. In addition to nicotine, the highly addictive stimulant in TS that provides smokers with instant gratification, TS contains many carcinogens generated during the curing and burning of tobacco leaves. However, E-cigarettes (E-cigs) were designed to deliver E-cig smoke (ECS), which comprises only nicotine in aerosolized gas phase solvents. E-cigs do not use tobacco leaves, and the generation of E-cig smoke (ECS) does not involve burning. Hence, ECS contains only nicotine and the gas phase solvents. E-cigs are promoted as delivering a TS `high' without the negative effects of TS. Given the rapid rise in the number of E-cig users, the health effects of ECS?particularly its carcinogenicity?deserves careful scrutiny. It is well established that during tobacco curing, the nitrosation of nicotine generates tobacco- specific nitrosamines including carcinogenic N'-nitrosonornicotine (NNN) and 4-(methylnitrosamine)-1-(3- pyridyl)-1-butanone (NNK). An important factor when investigating the carcinogenicity of ECS is determining whether nicotine nitrosation can occur in vivo. However, it can be difficult to measure NNN and NNK concentrations in humans and rodents as both have multiple cytochrome p450s that quickly transform NNK and NNN into methyldiazohydroxide (MDOH), formaldehyde (FAL), and pyridyl-butyl-derivatives (PBDs). As a result, the levels of NNK and NNN resulting from nitrosation of inhaled nicotine are very low at any given time. To circumvent this, we determine nicotine-induced DNA damage, rather than directly measuring the nicotine metabolites NNK and NNN. With our novel approach, it should be possible to detect DNA damage induced by the NNK and NNN metabolites, given that it takes hours to days for mammalian cells to repair DNA damage. Our recent results show that in human urothelial cells (HUCs), nicotine treatment: 1) induces mutagenic O6- methyl-deoxyguanosine and cyclic 1,N2-?-hydroxy-propano-dG adducts; 2) reduces DNA repair capacity; 3) downregulates DNA repair genes; and 4) enhances HUC's mutation and tumorigenic transformation. We also found that ECS induces DNA adducts in mouse bladder mucosa which cross react with PdG and O6-medG antibodies. Based on these results, we hypothesize that ECS is a bladder carcinogen that causes carcinogenesis through nitrosation of nicotine into NNN and NNK, and the metabolites can, in turn, damage DNA, modify DNA repair proteins, and enhance mutation susceptibility and tumorigenic transformation. We will test this hypothesis and determine the molecular mechanisms by using mouse models to determine: 1) ECS's effect on DNA repair in mouse bladder mucosa; 2) the chemical nature of ECS-induced DNA adducts; 3) the ECS-induced molecular alterations in mouse bladder tissues; and 4) the ECS bladder carcinogenicity. These studies will help dispel the unsubstantiated claim that ECS is safe and will discourage individuals from smoking E-cigs. Our proposed studies therefore are very timely and have extremely important public health implications.